Volume 18, Issue 1, Pages 25-31 (January 2011) How the Local Geometry of the Cu-Binding Site Determines the Thermal Stability of Blue Copper Proteins  Jesús Chaboy, Sofía Díaz-Moreno, I. Díaz-Moreno, Miguel A. De la Rosa, Antonio Díaz-Quintana  Chemistry & Biology  Volume 18, Issue 1, Pages 25-31 (January 2011) DOI: 10.1016/j.chembiol.2010.12.006 Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 1 Comparison between Pcs from Phormidium and from Synechocystis (A and B) Diagrams of (A) Pho-WT (pdb code: 1baw) and (B) Syn-WT (1pcs) Pcs (21, 22). Residues are colored according to conservation patterns from ConSurf phylogenetic computations, which include all the BCP family. Average residues are in white. Color scale corresponds to residues with increasing variability as compared to average. Red corresponds to highly conserved residues and blue to variable amino acids. ConSurf (Landau et al., 2005) calculations are unreliable for residues colored in yellow. Graphics are performed with UCSF Chimera (Pettersen et al., 2004). The arrow in (A) points to the location of the site-directed mutation. (C) Detailed view of the copper site. Residues within 5 Å from the metal ion are represented in sticks. Those containing first-sphere atoms are labeled. Backbone ribbon is colored as in (A) and (B). (D) Comparison of Tm values among Pc species (Feio et al., 2004, 2006; Muñoz-López et al., 2010a). Chemistry & Biology 2011 18, 25-31DOI: (10.1016/j.chembiol.2010.12.006) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 2 XANES Experiments The top panel shows a detailed comparison of the experimental XANES spectrum at the Cu K-edge of Pc in Syn-WT (●) and Pho-WT (○). The dotted blue line corresponds to the spectrum recorded in the case of Pho-Mut. In all the cases the zero energy has been taken as E0 = 8981eV. The bottom panel shows a comparison of the experimental XANES spectrum at the Cu K-edge in Syn-WT Pc (●) and the theoretical spectra (green, solid line) calculated by using a complex Dirac-Hara ECP potential within the two-channel approach (Chaboy et al., 2005, 2006a, 2006b). See also Figures S1 and S2. Chemistry & Biology 2011 18, 25-31DOI: (10.1016/j.chembiol.2010.12.006) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 3 XANES of the Oxidized Forms Comparison of the theoretical spectra calculated for Cu in Syn-WT (●) and Pho-WT (○) and those of: a 151 atom Pho-WT cluster in which the first 15 (blue, dots) and 113 (purple, dash) atoms have been substituted by the structural arrangement of Syn-WT (top panel); a 151 atom Pho-WT cluster in which the next neighbors associated to the histidines (blue, dots), cysteine (red, dash), and methionine (green, dot-dash) within the first 4 Å around the absorbing Cu have been substituted for those found in Syn-WT (middle panel); and of a 151 atom Pho-WT cluster in which the N atoms associated to the histidines (black, solid line, and red, dots), and the sulfur ones associated to cysteine (green, dash) and methionine (blue, dot-dash) have been substituted for those found in Syn-WT (bottom panel). Chemistry & Biology 2011 18, 25-31DOI: (10.1016/j.chembiol.2010.12.006) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 4 XANES of the Reduced Forms The top panel shows a detailed comparison of the experimental XANES spectrum at the Cu K-edge of the reduced Pc in Synechocystis (●) and Phormidium (○). The dotted blue line corresponds to the spectrum recorded in the case of Pho-Mut. The bottom panel shows a comparison of the theoretical spectra calculated for Syn-WT (○) and Pho-WT (○) by adopting the structural Cu environment of the oxidized forms and those in which the Cu-Sγ,Cys and Cu-Sδ,Met bond lengths are fixed to intermediate values between those found in the oxidized forms. Chemistry & Biology 2011 18, 25-31DOI: (10.1016/j.chembiol.2010.12.006) Copyright © 2011 Elsevier Ltd Terms and Conditions